Metabolic Solutions

Abstract Background and Aims CHK-336 is a first-in-class, orally bioavailable, liver-targeted small molecule lactate dehydrogenase (LDH) inhibitor for the treatment of primary hyperoxalurias and other kidney stone disorders caused by oxalate overproduction. The primary objectives of this first-in-human (FIH) study were characterization of the safety and pharmacokinetics (PK) of CHK-336 following single and repeat doses in healthy volunteers (HVs). Exploratory objectives were demonstration of proof of mechanism in HVs based on inhibition of hepatic oxalate production using a stable-label 13C2-glycolate tracer for hepatic LDH target engagement and characterization of the exposure-response relationship. Method This Phase 1, placebo-controlled (6:2 randomization within a cohort), dose escalation study explored single ascending (SAD, 15-500 mg) and multiple ascending (MAD, 30 mg and planned up to 500 mg QD, 14 consecutive days) doses. A total of 104 subjects will be enrolled in this study. A stable-label 13C2-glycolate tracer was administered orally at baseline and following CHK-336 administration to evaluate the inhibition of LDH-mediated production of [13C2]-oxalate by CHK-336, as a measure of hepatic LDH target engagement in HVs. Food-effect was evaluated following a single dose after a standard high fat meal. CHK-336 concentrations were determined in plasma and urine by a validated LC-MS/MS assay. Total oxalate and 13C2-oxalate were determined in urine by validated GC/MS assays. Results CHK-336 was generally well tolerated following single doses from 15 – 500 mg, with no dose-related trends in total adverse events (AEs) or types of AEs. There were no serious AEs, or severe treatment-related AEs (TRAEs). Treatment-emergent AEs were observed in 10 of 56 (17.9%) subjects, 7 (12.5%) of whom experienced TRAEs; all TRAEs were mild or moderate in severity. CHK-336 PK profiles were characterized with a median tmax of 6 hrs following absorption in fasted-state followed by bi-exponential decline and a terminal half-life of 18 ± 8 hrs. Dose-proportional increases in exposure AUC were observed from 15 up to 500 mg. Cmax increase was greater than dose-proportional at ≥60 mg with a concomitant decrease in median tmax from 6hr to 2hr. A high fat meal decreased CHK-336 AUC0-72h by 32% (AUC0-72h) and Cmax by 55%. Renal elimination of unchanged CHK-336 was quantitatively minor (<1% of administered dose). A direct effect inhibitory Imax-IC50 model adequately described the relationship between CHK-336 plasma concentrations and urinary 13C2-oxalate (% of control). Robust target engagement was demonstrated with an exposure-related reduction in urinary 13C2-oxalate from 15 – 60 mg, with maximum inhibition achieved at CHK-336 doses ≥60 mg. Conclusion CHK-336 is a first-in-class, orally administered, liver-targeted LDH inhibitor that was generally well-tolerated following single doses from 15 – 500 mg, with dose proportional increases in AUC exposure. Hepatic oxalate production was robustly reduced by CHK-336, as measured by urinary 13C2-oxalate, thus achieving proof of mechanism in HVs.

The synthesis of various benzo[h]quinazoline analogs was accomplished through the reaction of chalcone with guanidine. The synthesized compounds were screened for their anticancer potential against different cancer cells.

We recently demonstrated the ability to simultaneously quantify lipid and protein flux in vivo using oral D2O dosing. The uniqueness of using D2O is the rapid equilibration with total body water, use of oral and not intravenous dosing, and ease to measure the true precursor enrichment (either plasma, urine, or saliva). To examine whether dietary modulation of de novo fatty acid and cholesterol synthesis would lead to parallel changes in apoB and/or A1 production, non‐human primates were either fed a standard carbohydrate‐based diet or a high‐fat, high‐cholesterol diet for several months. Lipid and protein kinetics were studied by giving a single oral bolus of D2O and sampling blood for ~ 96 hours thereafter. The dietary perturbation altered the plasma lipid profiles. The analyses of lipid labeling (measured via GC‐IRMS) demonstrated a marked suppression of fatty acid and cholesterol synthesis with high‐fat feeding. Despite these changes we observed a substantial increase in both apoB and A1 synthesis (measured via LC‐MS). In total, we demonstrate that diet‐induced modulation of pathways involving de novo lipid synthesis do not necessarily translate into a comparable protein‐flux phenotype. Our data suggest that the dietary lipid, as compared to de novo synthesized lipid, has a pronounced effect on circulating apo‐protein flux. The methodology applied here can be readily adapted for studies in other pre‐clinical models (e.g. rodents) as well as applied in humans. This protocol design provides a translational biomarker approach for interrogating modulations that affect dyslipidemia.

Postprandial high glucose and insulin responses after starchy food consumption, associated with an increased risk of developing several metabolic diseases, could possibly be improved by altering food structure. We investigated the influence of a compact food structure; different wheat products with a similar composition were created using different processing conditions. The postprandial glucose kinetics and metabolic response to bread with a compact structure (flat bread, FB) was compared to bread with a porous structure (control bread, CB) in a randomized, crossover study with ten healthy male volunteers. Pasta (PA), with a very compact structure, was used as the control. The rate of appearance of exogenous glucose (RaE), endogenous glucose production, and glucose clearance rate (GCR) was calculated using stable isotopes. Furthermore, postprandial plasma concentrations of glucose, insulin, several intestinal hormones and bile acids were analyzed. The structure of FB was considerably more compact compared to CB, as confirmed by microscopy, XRT analysis (porosity) and density measurements. Consumption of FB resulted in lower peak glucose, insulin and glucose-dependent insulinotropic polypeptide (ns) responses and a slower initial RaE compared to CB. These variables were similar to the PA response, except for RaE which remained slower over a longer period after PA consumption. Interestingly, the GCR after FB was higher than expected based on the insulin response, indicating increased insulin sensitivity or insulin-independent glucose disposal. These results demonstrate that the structure of wheat bread can influence the postprandial metabolic response, with a more compact structure being more beneficial for health. Bread-making technology should be further explored to create healthier products.

Low‐grade inflammation is associated with insulin resistance and chronic postnatal overnutrition (CPO) results in a rapid increase in body weight, hyperinsulinemia and hyperleptinemia. We tested whether CPO would increase susceptibility to the development of insulin resistance in response to HFD by further enhancing the inflammatory response. Swiss Webster pups were raised in either 3pups/litter (CPO) or 10pups/litter (CTR) until weaning. At 6 wks of age, a subset of mice was placed on a HFD. CPO mice were significantly heavier and the body weight difference was exacerbated by HFD. While all HFD mice had increased basal glucose levels and decreased glucose clearance during GTTs, CPO‐HFD had significantly higher glucose levels than all other groups. F4/80 immunohistochemistry in eWAT was evident in HFD animals, but was significantly higher in the CPO‐HFD group. HFD increased the gene expression of several inflammatory cytokines, TNFα, IL‐6 and macrophage migration inhibitory factor (MIF), and decreased gene expression of anti‐inflammatory adipokine adiponectin, with the change being much greater in the CPO‐HFD group. Overall, these data provide the evidence that postnatal exposure to overnutrition results in abnormalities in macrophages and adipocytes in WAT, which likely increases susceptibility to insulin resistance and exacerbates the detrimental outcome in response to HFD in adulthood.

Objective: The aim of this work was to determine if dysprosium chloride (DyCl3) is a suitable nonabsorbable marker for studies of labeled-triglyceride excretion in cystic fibrosis patients allowing excretion to be determined accurately after analysis of one or two stools.Methods: A series of 66 absorption studies were conducted in 36 young cystic fibrosis patients over a five year period. All tests consisted of ingesting a single test meal containing both 13C-labeled triglyceride (TG*) and DyCl3; in most studies the food colorant brilliant blue (FD&C blue #1) was administered along with the DyCl3. Ingestion of the test meal was followed by collection of individual stools for 72 to 96 hours. Stools were analyzed for 13C-Excess (13C*) and Dy.Results: Excretion of Dy in cystic fibrosis patients who exhibited a wide-range of steatorrhea was quantitative. Fractional excretion of Dy and 13C* in individual stools showed a high linear correlation (r2 = 0.969) with a slope and y-intercept close to unity and zero, respectively. As a result, estimates of TG* excretion based on analysis of only two stools (partial pool method, PPM) were not different from those based on the analysis of all stools or stool composites. This was true both when Dy content and when stool color due to ingested brilliant blue was used to determine which stools to analyze for the PPM.Conclusions: Combining the use of Dy and brilliant blue permits reasonably accurate estimates of fecal TG* excretion after analysis of samples from two easily identified stools. This practical method can be used to address many important clinical and experimental questions regarding triglyceride digestion and absorption that may otherwise go unanswered.

Our purpose was to determine the effect of meal size on gastric emptying (GE) as measured by octanoate breath test (OBT), to determine the effect of the duration of breath collections on assessment of GE by OBT, and to determine the effect of meal size on gastric myoelectrical activity as measured by electrogastrography (EGG). Fourteen normal subjects underwent two modified [13C]OBTs using muffin meals of 250 or 350 kcal mixed with 100 mg [13C]sodium octanoate. T(1/2) for GE was determined for both the entire postprandial 6-hr breath collection and a truncated initial 4-hr data set. EGG was recorded for 30 min prior to the muffin meal and 4 hr postprandially. Using the 6-hr breath collection data, the T(1/2) was 177 +/- 7 (mean +/- SEM) for the 350-kcal meal compared to 153 +/- 7 min (P < 0.01) for the 250-kcal meal. Using the 4-hr data, the T(1/2) for the 350-kcal meal was 244 +/- 32 min compared to 165 +/- 12 min (P < 0.05) for the 250-kcal meal. The ratio of postprandial to fasting EGG power of the dominant frequency for the 350-kcal meal (1.9 +/- 0.4) was higher than that for the 250-kcal meal (1.3 +/- 0.6). T(1/2) for the 350-kcal meal using 4- and 6-hr data was significantly correlated with the 4-hr power ratio (r = 0.68 and 0.67; P < 0.05, respectively), but poorly correlated for the 250-kcal meal. In conclusions, GE and EGG are affected by meal size. Using the muffin-based [13C]OBT, T(1/2) for the 350-kcal meal was significantly longer than for a 250-kcal meal. Longer T(1/2) values were obtained with shorter breath sampling durations. The postprandial to fasting power ratio for the 350-kcal meal was greater than that for the 250-kcal meal.

Microvesicular steatosis is a potentially severe manifestation of valproic acid hepatotoxicity due to an acquired impairment of mitochondrial function. Tests currently used to investigate mitochondrial fonction are cumbersome and cannot be used routinely. We report on a noninvasive breath test using [13C]methionine for estimation of mitochondrial function in a case of pure severe valproic acid overdose-induced microvesicular changes. The initially abnormal breath test improved together with the recovery of liver failure. In conclusion, this observation suggests that the [13C]methionine breath test may provide a noninvasive estimate of hepatic mitochondrial function in vivo.

Stable isotope labeling in therapeutic and subtherapeutic quantities of drug (15N2(13)C-phenobarbital) can be quantitated in biological matrices (urine) and high performance liquid chromatography (HPLC) peaks from urine using continuous-flow isotope-ratio mass spectrometry (CF-IRMS). Standard curves for 15N2(13)C-phenobarbital were reproducible and linear (R2 > 0.985) over the ranges of 3-100 micrograms/ml for whole urine (15N2 or 13C labeling) and 0.1-8.0 micrograms/mL for HPLC peaks derived from urine (15N2 labeling). The lower limit of quantitation values for urine drug concentration was 0.46-2.62 micrograms/mL in whole urine and 0.10-0.70 micrograms/mL in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. These data suggest stable isotope labeling and CF-IRMS may be used as an alternative to 14C labeling and radioactivity counting methods in mass balance/metabolite identification and other biomedical studies.